Immunogenic conjugates comprising a group B meningococcal porin and an H. influenzae polysaccharide

ABSTRACT

Disclosed is an  H. influenzae  type b polysaccharide-meningococcal outer membrane protein conjugate, pharmaceutical compositions thereof, and the use thereof to induce an immune response to  H. influenzae  in an animal.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention is in the field of vaccines useful for raising an immune response in an animal. In particular, the invention relates to H. Influenzae polysaccharide-N. miningitidis outer membrane protein conjugates, pharmaceutical compositions and the use thereof.

[0003] 2. Background Information

[0004]Haemophilus influenzae are small, pleomorphic, Gram-negative coccobacilli. Isolates are classified into six antigenically distinct capsular types (a-f) and nonencapsulated, nontypable strains. Haemophilus influenzae can cause meningitis, otitis media, sinusitis, epiglottitis, septic arthritis, occult febrile bacteremia, cellulitis, pneumonia, and empyema; occasionally this organism causes neonatal meningitis and septicemia. Other H. influenzae infections include purulent pericarditis, endocarditis, conjunctivitis, osteomyelitis, peritonitis, epididymo-orchitis, glossitis, uvulitis, and septic thrombophlebitis. Most cases of invasive diseases in children before the introduction of H. influenzae type b (Hib) conjugate vaccination were caused by type b. Nonencapsulated organisms can cause invasive disease in newborns. Nonencapsulated strains cause upper respiratory tract infection, including otitis media, sinusitis, and bronchitis, and may cause pneumonia.

[0005] The source of the organism is the upper respiratory tract of humans. The mode of transmission is presumably person to person, by direct contact, or through inhalation of droplets of respiratory tract secretions containing the organism. Asymptomatic colonization by nonencapsulated strains is frequent; organisms are recovered from the throat of 60% to 90% of children. Colonization by type b organisms, however, is infrequent, ranging from 2% to 5% of children in the pre-vaccine era, and appears to be even less frequent with widespread Hib conjugate vaccination. The exact period of communicability is unknown but may be for as long as the organism is present in the upper respiratory tract.

[0006] Before the introduction of effective vaccines, Hib was the most common cause of bacterial meningitis in children in the United States and in many other countries. Meningitis and other invasive infections were most common in children 3 months to 3 years of age and approximately half of the cases occurred in infants younger than 12 months. The age-specific incidence of invasive type b disease in different populations in countries has varied; the proportion of disease in infants younger than 12 months tends to be greatest in populations with the highest total incidence, resulting in a lower median age of cases. In contrast to meningitis and most other invasive Hib diseases, epiglottitis is rare in infants younger than 12 months; its peak occurrence in the pre-vaccine era was 2 to 4 years of age. Epiglottitis also can occur in older, unvaccinated children and adults.

[0007] Invasive disease has been more frequent in boys, African-Americans, Alaskan Eskimos, Apache and Navajo Indians, child care center attendees, children living in overcrowded conditions and children who were not breast-fed. Unimmunized children, particularly those younger than 4 years who are in prolonged, close contact (such as in a household) with a child with invasive Hib disease, are at an increased risk for serious infection from this organism. Other factors predisposing to invasive disease include sickle cell disease, asplenia, HIV infection, certain immunodeficiency syndromes, and malignancies. Infants younger than 1 year with documented invasive infection are at an approximate 1% risk of recurrence, if not subsequently vaccinated.

[0008] Since 1988 when Hib conjugate vaccines were introduced, the incidence of invasive Hib disease has declined by 95% in infants and young children and the incidence of invasive infections caused by other encapsulated types is now similar to that caused by type b. As a result of this success, the U.S. Public Health Service has targeted Hib disease in children younger than 5 years for elimination in this country. Invasive Hib disease occurs now in this country primarily in under vaccinated children and among infants too young to have completed the primary series of vaccinations.

[0009] Four Hib conjugate vaccines have been licensed in the United States. These vaccines consist of the Hib capsular polysaccharide (ie, polyribosylribotol phosphate [PRP] or PRP oligomers) covalently linked to a carrier protein directly or via an intervening spacer molecule. Protective antibodies are directed against PRP. Conjugate vaccines differ in composition and immunogenicity and, as a result, recommendations for their use differ. For example, PRP-D is recommended only for children 12 months of age and older, whereas the other three vaccines, HbOC, PRP-T, and PRP-OMP, are recommended for infants beginning at 2 months of age.

[0010] Adjuvants are substances that augment the immune response to antigens and, therefore, have been used in many vaccines and vaccine candidates. The immune stimulatory effect of adjuvants is not antigen specific, as they boost immune responses towards many different types of antigens. The only adjuvants currently approved for human use by the FDA are aluminum salts, but many adjuvants used in animal vaccinations and in newer vaccine candidates are microbial in origin (61) e.g. Freund's adjuvant, Corynebacterium parvum, muramyl dipetide, tetanus toxoid, etc. The mechanisms for the immunopotentiating ability of microbial substances are unknown.

[0011] The major outer membrane proteins of the pathogenic Neisseria (Neisseria gonorrhoeae and Neisseria meningitidis) have been investigated for adjuvant potential (36, 37, 39, 40, 60) and for the mechanism behind their immunopotentiating ability. The proteins of interest are protein IA (PIA) and protein IB (PIB) from the gonococcus and class 1, 2 or 3 proteins from the meningococcus (C1, C2 and C3 respectively) (4). They all function as porins (41, 43, 62), have significant amino acid sequence homology amongst each other (6, 7, 21, 59) and are considered to be part of the gram negative porin superfamily (26).

[0012] Neisserial porins, when complexed non-covalently with malarial peptides, were shown to enhance the antibody response to these peptides as compared to when the peptides were used as an immunogen alone or covalently linked to other proteins (39, 40). In addition, peptides derived from Group A streptococcus (38), influenza virus hemagglutinin (38), or Trypanosome bruceei (40) were shown to be more immunogenic in mice when incorporated into complexes containing Neisserial porins as compared to when the mice were immunized with peptides alone. Meningococcal outer membrane vesicles (OMV), mainly consisting of the class 2 protein, were used as a carrier to boost the immune response towards the H. influenzae polysaccharide capsule in the recently licensed H. influenzae type b vaccine developed by Merck (10). Furthermore, Livingston has explored the use of purified Neisserial porins as adjuvants in anti-melanoma vaccines. Melanoma cells express much higher levels of the human gangliosides GM2 or GD3 on their surface as compared to normal melanocytes. To augment the immune response to GM2 and GD3, and possibly induce tumor immunity in melanoma patients, GM2 and GD3 were noncovalently associated with purified Neisserial porins and volunteers with malignant melanoma were immunized with these vaccine constructs. Anti-GM2 or anti-GD3 antibody responses were greatly enhanced in patients immunized with porin/GM2 or porin/GD3 complexes as compared to patients immunized with these gangliosides alone or complexed with BCG (36, 37). In addition, the tumor burden in patients immunized with porin/GM2 decreased significantly (personal communication, P. Livingston).

[0013] The mechanisms by which the Neisserial porins act as adjuvants are unknown. The group from Merck (10, 35, 56), who developed the Haemophilus polysaccharide capsule—meningococcal OMV conjugate vaccine, thought that it might be due to direct T cell stimulation by the class 2 protein. They initially demonstrated that the class 2 protein could directly stimulate T lymphocytes and, therefore, they renamed the class 2 protein as the Meningococcal Immune Enhancing Protein (MIEP) (35). However, it was later shown that only denatured class 2 protein at high concentrations (>50 μg) could stimulate T cells, whereas the native protein had no such effect (56). Furthermore, since the majority of the Neisserial porins are in their native configuration when used as a vaccine candidate or adjuvant, the likelihood that non-specific T cell stimulation by denatured porins accounts for their immunopotentiating ability is low.

[0014] Over the last few years, details regarding the interaction between T and B lymphocytes required for antigen recognition, lymphocyte stimulation and antibody production have been elucidated. In the current model of T lymphocyte stimulation, two sets of signals between the antigen presenting cell (APC) and the T lymphocyte have been shown to be required (24, 25, 51). The first signal (signal 1) is delivered via the interaction of the major histocompatibility (MHC) complex on antigen presenting cells (e.g. B lymphocytes, dendritic cells, macrophages, etc.) and the T cell receptor on T lymphocytes. The groove on the MHC complex is usually occupied by an oligopeptide derived from processed antigens (T cell epitope). The specificity of the reaction is conferred by signal 1. The second or costimulatory signal (signal 2) is delivered by the binding of two sets of counter-receptors during the interaction between the B and T lymphocytes (FIG. 1). The activated T lymphocytes then release cytokines which in turn stimulate the effector cells, for example, causing B lymphocytes to become antibody producing cells. The induction of costimulation by the interaction of these counter-receptors has been shown to be important in tumor immunity (1, 3, 8, 11, 51, 55), the prevention of tolerance (19, 45, 54), and for cytotoxic lymphocyte activity (1).

[0015] The T lymphocyte counter-receptors are CD28 and CTLA-4. They are both members of the immunoglobulin superfamily (9). CD28 is present on resting and activated T cells (1, 8, 27, 30, 32, 34, 46), while CTLA-4 is only expressed on activated T cells (17, 23, 31, 33, 51). The level of CD28 on activated T cells is 20× higher than CTLA-4 but the affinity of CD28 for its B cell counter-receptor is much lower (31, 33). The B lymphocyte counter-receptors are B7 (14, 20, 32, 48, 49) and the more recently discovered B7-2 (2, 12, 13, 16, 24). B7 and B7-2 are members of the immunoglobulin superfamily (13-15) and are only present on activated B lymphocytes (14). Several lines of evidence demonstrate the relationship of the newer ligand, B7-2, to T lymphocyte costimulation; 1) CTLA-4 binding to activated B cells is only partially inhibited by an anti-B7 monoclonal antibody (mAb) (24), 2) lymphocytes derived from mice deficient in B7 expression can still costimulate T cells (12, 13), 3) transfectants expressing B7-2 alone can costimulate T cells (13, 16), and 4) a mAb specific for B7-2 can inhibit T lymphocyte costimulation by B cells (24) or B7-2 transfectants (13). The significance of the initially described B7 antigen as a costimulation counter-receptor is controversial because the expression of B7-2 occurs earlier than the expression of B7 and there is more B7-2 present on the surface of activated B lymphocytes than B7 (24). A schematic representation of T lymphocyte costimulation and the costimulatory counter-receptors is illustrated in FIG. 1.

[0016] There is preliminary evidence presented by various investigators that microbial products can stimulate B lymphocytes. Liu et al. have demonstrated that lipopolysaccharide (LPS), mitogenic influenza virus, and an antigen that mimics viral infection (polyinosinic-polycytidylic acid), all stimulate B lymphocytes, which in turn costimulate T lymphocytes (25). Vordermeier has demonstrated that purified Salmonella typhi porins (free of LPS) are potent B cell stimulators, but have minimal effect on T lymphocytes (57, 58). In addition, meningococcal outer membrane preparations, mainly consisting of the meningococcal porins act as B cell mitogens and do not stimulate T lymphocytes (44, 52, 53). This evidence suggests that Neisserial porins, and possibly other gram-negative porins, might be able to stimulate B lymphocytes and increase B7-2 expression The increased expression of B7-2 can mediate T lymphocyte costimulation and this could be a mechanism by which porins enhance the immune response to other antigens, such as the PRP polysaccharide presented here.

BRIEF SUMMARY OF THE INVENTION

[0017] The present invention relates to an H. influenzae type b (Hib) polysaccharide-substantially pure, refolded meningococcal outer membrane protein (rPorB) conjugate.

[0018] The present invention also relates to a method of preparing an Hib polysaccharide-rPorB conjugate, comprising

[0019] (a) obtaining an Hib polysaccharide,

[0020] (b) oxidizing or selectively hydrolyzing said polysaccharide to generate aldehyde groups;

[0021] (c) obtaining a rPorB; and

[0022] (d) conjugating the polysaccharide containing aldehyde groups to the rPorB by reductive amination.

[0023] The present invention also relate to the conjugates obtained according to the methods of the invention. Optionally, the conjugates of the present invention may be combined with DTaP (diphtheria, tetanus, acellular pertussis vaccine).

[0024] The present invention also relates to pharmaceutical compositions comprising the conjugates of the invention, optionally comprising DTaP, and a pharmaceutically acceptable carrier.

[0025] The present invention also relates to a method of inducing an immune response in an animal to H. influenzae, comprising administering the conjugates of the invention to the animal in an amount effective to induce said immune response.

[0026] The invention relates in part to the surprising discovery that the Hib-rPorB conjugates of the invention induce substantially greater immune responses in animals compared to when tetanus toxoid and the recombinantly produced outer membrane P2 protein from H. influenzae is used as the antigenic protein. Substantially greater immunogenic responses were also obtained compared to the Hib-CRM conjugate which is commercially available from Lederle Laboratories, Division of American Cyanamide Company, Pearl River, N.Y. CRM₁₉₇ is a site mutant, non-toxic variant of diphtheria toxin isolated from cultures of Cornebacterium diphtheriae C7(β197). Seid, R. C. Jr. et al., Glycoconj. J 6: 489-498 (1989).

[0027] Furthermore, the conjugate of the present invention is especially useful in compositions also comprising DTaP, as immunologic interactions between the components, as well as epitopic suppression, is observed with conventional carrier proteins such as tetanus toxoid. The conjugates of the present invention overcome this serious limitation in combination vaccine compositions.

BRIEF DESCRIPTION OF THE FIGURES

[0028]FIG. 1 depicts a graphical representation of T-lymphocyte costimulation.

[0029]FIG. 2 depicts a graph showing B1HB 1030 fermentation profile.

[0030]FIG. 3 depicts a stained SDS-PAGE gel of the purified rPorB used for conjugation.

[0031]FIG. 4 depicts a bar graph showing the Hib polysaccharide-specific IgG response in rats for Hib conjugates with various carrier proteins.

[0032] FIGS. 5A-5G depict tables showing serum antibody of Sprague Dawley rats immunized with Hib-TT, Hib-rPorB and Hib-rP2 measured by ELISA.

[0033]FIGS. 6A and 6B depict data showing PRP-specific ELISA IgG response in rats for Hib conjugate vaccines with different carrier proteins. Two different Hib-rPorB preparations were tested (−1 and −2). FIG. 6A is a graphical depiction of the tabular data shown in FIG. 6B.

[0034]FIG. 7 depicts a graph showing polysaccharide-specific IgG elicited by Hib conjugate vaccines in CD-1 mice.

[0035]FIG. 8 depicts a graph showing polysaccharide-specific IgG elicited by Hib conjugate vaccines in rats.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention relates to a vaccine for inducing an immune response in an animal comprising the outer membrane meningococcal group B porin protein linked to Hib polysaccharide, together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the vaccine may be administered in an amount effective to elicit an immune response in an animal to H. influenzae. In a preferred embodiment, the animal is a mammal selected from the group consisting of humans, cattle, pigs, sheep, and chickens. In another preferred embodiment, the mammal is a human.

[0037] By the term “rPorB” is intended the mature, refolded class 2 or class 3 outer membrane protein from N. meningitidis and fusions thereof comprising amino acids 1 to 20 or 1 to 22 of the T7 gene φ10 capsid protein. Methods for high level expression of the mature class 2 and class 3 rPorB and fusions thereof, refolding, and purification are described in (47) and U.S. Pat. No. 5,439,808, the disclosures of which are incorporated herein by reference. The recombinant porin may be expressed at high levels from E. coli according to U.S. Pat. No. 5,439,808, or from yeast according to Ser. No. 08/92,302. In a preferred embodiment, the class 3 rPorB is expressed from the host BL21(DE3)ΔompA which has been transformed with the gene coding for the rPorB, is substantially pure and is refolded according to U.S. Pat. No. 5,439,808.

[0038] The H. influenzae capsular polysaccharide can be isolated according to methods well known to those of ordinary skill in the art. See, Schneerson et al., J. Exp. Med. 152:361-376 (1980); Marburg et al. J. Am. Chem. Soc. 108:5282 (1986); Jennings et al., J. Immunol. 127:1011-1018 (1981); and Beuvery et al., Infect. Immunol. 40:39-45 (1983). In a preferred embodiment, the organism is cultured, the culture supernatant is microfiltered, and the filtrate is passed through a 300,000 molecular weight cut off filter. The permeate is then concentrated, for example, with a 100,000 molecular weight cut off filter. This 100,000-300,000 molecular weight material is then oxidized with a mild oxidant such as metaperiodate, the product filtered through a 30,000 molecular weight filter and then concentrated with a 5,000 molecular weight filter, to give a polysaccharide having aldehyde groups that may be used directly for conjugation. The preferred polysaccharide has a molecular weight of about 5,000-50,000. A more preferred polysaccharide has a molecular weight of about 10,000-50,000, however other molecular weight ranges may be employed as desired.

[0039] It will be understood by those of skill in the art that the capsular polysaccharide-protein carrier conjugates of the vaccine may be produced by several different methods. The types of covalent bonds which couple a polysaccharide to a protein carrier, and the means of producing them, are well known to those of skill in the art. Details concerning the chemical means by which the two moieties can be linked may be found in U.S. Pat. Nos. 5,623,057, 5,371,197, 5,192,540, 4,902,506 and 4,356,170, the contents of which are herein incorporated by reference in their entirety. For a review, see Contributions to Microbiology and Immunology, vol 10, Conjugate Vaccines, volume editors J. M. Cruse and R. E. Lewis, Jr., 1989, and (29). One such method is the reductive amination process described in Schwartz and Gray (Arch. Biochim. Biophys. 181:542-549 (1977)). This process involves producing the polysaccharide in a form which has reducing end groups, and reacting the capsular polysaccharide and rPorB in the presence of cyanoborohydride ions, or another reducing agent. The reducing groups may be formed by selective hydrolysis or specific oxidative cleavage, or a combination of both.

[0040] The vaccine of the present invention comprises the Hib-rPor conjugate, in an amount effective depending on the route of administration. Although subcutaneous or intramuscular routes of administration are preferred, the meningococcal group B porin protein, fusion protein or vaccine of the present invention can also be administered by an intraperitoneal, intravenous, or intranasal route. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Suitable amounts are expected to fall within the range of 5 to 50 μg per animal, more preferably, about 10 μg per animal.

[0041] The vaccine of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the conjugate vaccine has suitable solubility properties. The vaccines may be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination programs. Reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md. (1978), for methods of preparing and using vaccines.

[0042] The vaccines of the present invention may further comprise adjuvants which enhance production of H. influenzae-specific antibodies. Such adjuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant (CFA), stearyl tyrosine (ST, see U.S. Pat. No. 4,258,029), the dipeptide known as MDP, saponin, aluminum hydroxide, and lymphatic cytokine.

[0043] Freund's adjuvant is an emulsion of mineral oil and water which is mixed with the immunogenic substance. Although Freund's adjuvant is powerful, it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) or ST may be used for administration to a human. The conjugate vaccine may be absorbed onto the aluminum hydroxide from which it is slowly released after injection. The conjugate vaccine may also be encapsulated within liposomes according to Fullerton, U.S. Pat. No. 4,235,877.

[0044] In another preferred embodiment, the conjugate of the invention is combined with other immunogens that are used to vaccinate animals. Thus, the conjugate of the invention may be combined with DTaP or DTaP IPV for administration to the animal. DTaP is a combination vaccine for diphtheria, tetanus, and acellular pertussis, which is available from Amvax, Inc., Beltsville, Md. In a preferred embodiment, the acellular pertussis is in an oxidized form as is available from Amvax, Inc.

[0045] In another preferred embodiment, the present invention relates to a method of inducing an immune response in an animal comprising administering to the animal the vaccine of the invention in an amount effective to induce an immune response. Optionally, the vaccine of the invention may be coadministered with effective amounts of other immunogens as mentioned above to generate multiple immune responses in the animal.

[0046] The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in this art which are obvious to those skilled in the art are within the spirit and scope of the present invention.

EXAMPLES Example 1 rPorB Expression, Isolation, Refolding, and Purification

[0047] Bacterial strains, growth conditions, and reagents—Genomic DNA was isolated from Group B N. miningitidis strain 44/76 (serotype 15) employing standard procedures and used as a polymerase chain reaction template for amplification of the class 3 protein gene as described elsewhere (47). The amplified product was cloned into NdeI and Xhol sites of the pET17b plasmid (Novagen, Inc.) that was used to transform competent E. coli DH5α. Plasmid DNA from selected DH5α clones was isolated and employed to transform E. coli BL21 [DE3]-ΔompA. The transformants were selected by carbenicillin and expression induced by addition of IPTG to a final concentration of 0.4 mm.

[0048] Overexpression of rPorB in E. coli and refolding purification procedures. The levels of rPorB protein expressed at various times after induction were monitored by subjecting the cell extracts to SDS-PAGE in 8-16% gradient gels, using a Novex system (Novex, San Diego, Calif.) followed by Comassie brilliant blue staining, and densitometric analysis using a Digital Imaging System, model IS-1000 (Alpha Innotech Co., San Leandro, Calif.). The overexpressed rPorB was isolated by resuspending and lysing the bacterial cells with a Stansted air-driven cell disrupter (Stansted Fluid Power Ltd.) in TEN buffer (50 mm Tris-HCl, 1 mm EDTA, 100 mm NaCl, pH 8.0) followed by centrifugation and isolation of the pellet containing rPorB aggregated in the form of inclusion bodies (IBs). After washing the pellet with 0.5% deoxycholate in TEN buffer, followed by two rinses with TEN buffer, the protein was solubilized by resuspending and sonicating the IBs in freshly prepared 8 M urea solution for 5 min using a water bath sonicator. Refolding of rPorB into its native conformation was achieved by employing a detergent-assisted refolding procedure. Equal volumes of urea-dissolved IBs and 10% Z 3-14 (Calbiochem) were combined and the final porin extract applied to a Sephacryl S-300 (5×100 cm) column (Pharmacia Biotech Inc.) equilibrated in a buffer comprised of 100 mm Tris-HCl, 200 mm NaCl, 10 mm EDTA, 20 mm CaCl₂, and 0.05% Z 3-14, pH 8.0. Fractions containing rPorB were identified by SDS-PAGE, pooled, and applied to a Hiload Q-Sepharose HP ion exchange (2.6×20 cm) column (Pharmacia) equilibrated in 25 mm Tris-HCl, 200 mm NaCl, 1.0 mm EDTA, and 0.05% Z 3-14 pH 8.0. A gradient of 0.2-1.0 mm NaCl was applied and rPorB eluted as a single peak. Protein concentration was estimated by measuring the absorbance at 280 nm, employing a HP Model 8453 UV/V is rapid scan spectrophotometer equipped with a diode array detector (Hewlett-Packard Company, Palo Alto, Calif.), using a molar extinction coefficient of 41,960 which was calculated based on PorB aromatic amino acid content according to Mach et al. (42).

Example 2 PRP Polysaccharide Production, Purification and Oxidation

[0049] A 14 L fermentation of H. influenzae type B was performed using MAE II media as follows. H. influenzae type B strain Eagan was obtained in a 4 mL seed culture vial from the Ultra Low liquid nitrogen freezer and thawed at room temperature for thirty minutes. A 250 mL shake flask with 50 mL of MME II medium (10 mg/L hemin) was inoculated with 1 mL of seed culture to produce seed 1 (SI). The SI flask was incubated for 10 hours at 37° C. and 150 RPM in a shaker incubator (Innova 4330, New Brunswick Sci.). Twelve mL of SI were used to inoculate 600 mL of MME II (10 mg/1-hemin) in a 2.8 L Fernbach flask to produce SII. The SII flask was incubated for 9 hours at 37° C. and 150 RPM in a shaker incubator. Six hundred mL [4% (v/v) inoculum] of SII culture were used to inoculate 13.4 L of MME II (10 g/L xylose, 10 mg/L hemin) in a 20 L BIOFLO IV fermentor (New Brunswick Sc.). An example of the fermentation profile is shown in FIG. 2. After 10 h of the fermentation, harvesting by microfiltration was initiated, using a hollow fiber cartridge with a 0.2 μm pore size rating with a 0.14 m² surface area made out of polysulfone (Milipore). The permeate was sterile filtered into a 20 L carboy and the carboy was placed at 2-8° C. until further processed. The filtrate was then processed through a 300,000 molecular weight cut off (MWCO) filter (Milipore) and the permeate retained. This permeate was then applied to a 100,000 (MWCO) filter (Milipore) and concentrated to greater than 20 mg/ml. The retentate was oxidized at 25° C. for 2 h with sodium metaperiodate. The oxidized PRP was ultra filtrated through 30,000 MWCO filter (Milipore) and the permeate retained. This permeate was then applied to a 5,000 MWCO filter (Milipore), concentrated to a final concentration of greater than 90 mg/ml, diafiltrated against DI water, and lyophilized.

Example 3 PRP-PorB Conjugate Preparation

[0050] The purified rPorB used for conjugation is shown in FIG. 3. The previously described oxidized PRP polysaccharide was added to a rPorB solution (at 10 mg/ml concentration in 0.25 M HEPES, 0.2 M NaCl, and 0.05% Zwittergen 3, 14 of pH 8.5) to make a 10 mg/ml polysaccharide solution. The solution was mixed for 1 min after which sodium cyanoborohydride was added to a final concentration of 6 mg/ml. The solution was then place in a water bath at 28-30° C. for 16 to 24 h. The conjugation reaction was stopped by the addition of 2 M ethanolamine solution at pH 8.5 and incubated at 28 to 30° C. for additional 16 to 24 hours. The reaction mixture was then applied to a Superdex 200 prep Grade column (Pharmacia) preequilibrated and run with PBS containing 0.01% thimerosal. The fraction eluting in the void volume of this column as monitored by UV-280 nm absorbance were collected, pooled, and stored at 4° C. prior to their analysis. Two chemical analysis were preformed to assess the PRP content (orcinol/ferric chloride/hydrochloric acid assay (50)) and the rPorB content (Coomassie protein assay (5)).

Example 4 Evaluation of the PRP-rPorB Conjugate in Rats

[0051] Female Sprague-Dawley rats (4-6 weeks old) in groups of ten were injected subcutaneously with 10 μg of conjugated PRP in 0.5 ml of PBS containing 0.01% thimerosal, either unabsorbed or preabsorbed on aluminum hydroxide (Alhydrogel, Superfos, Denmark) (final elemental aluminum concentration of 1 mg/ml) at days 0, 28, and 49. Bleeds were performed at day 0, 28, 38, 49, and the animals exsanquinated at day 59.

[0052] Serum antibody measurement by ELISA. The human serum albumin (HSA) (Sigma, St. Louis, Mo.) conjugates used for ELISA assays were prepared by reductive amination as previously described. The oxidized PRP polysaccharide was added to HSA followed by reduction with NaBH₃CN as described (28). The conjugates were isolated by gel filtration chromatography, and stored freeze-dried at −70° C. PRP-specific antibody titers were determined by an enzyme-linked immunosorbent assay (ELISA). Polystyrene, 96-well, flat-bottom microtiter plates (NUNC Polysorb) (Nunc, Naperville, Ill.) were coated with PRP-HSA conjugates in PBS (0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4) at 0.25 μg/well (100 μl/well) by incubating for 1 hour at 37° C., followed by a PBS-Tween (0.05% [v/v] Tween 20 in PBS) wash (5 times). All subsequent incubations were conducted at room temperature. PBS-Tween was used for all required washes. The coated plates were then blocked with PBS and 0.1% (w/v) Carnation nonfat dry milk for IgM ELISAs at 0.15 ml/well for 1 hour, followed by a wash. Sera were diluted 2-fold, in duplicate, in the plate at 100 μl/well and incubated for 1 hour, followed by a wash. Antibody conjugate (peroxidase-labelled goat anti-rat [Kirkegaard & Perry Lab, Gaithersburg, Md.] was added at 100 μl/well and incubated for 30 minutes, followed by a wash. A 1:1 dye and substrate solution (Kirkegaard & Perry TMB and peroxide) was added at 0.05 ml/well and incubated for 10 minutes. The peroxidase reaction was then stopped with 1M H₃PO₄ at 0.05 ml/well, and the plate was read on a Molecular Devices Emax microplate reader (Molecular Devices, Menlo Park, Calif.) at a wavelength of 450 nm, using 650 nm as a reference wavelength. Background absorbances were determined in several no-serum control wells and averaged for each plate. For each serum dilution, the average background absorbance was subtracted, and then duplicate serum absorbance values were averaged. A modified Scatchard plot was used for the subsequent data analysis, where the absorbance (y-axis) was plotted against the absorbance times the reciprocal dilution (x-axis) (18, 22). Under conditions allowing equilibrium and antibody excess, a straight line was obtained for each serum dilution series; this line was extrapolated to the x-axis for the determination of an antibody titer. A positive control serum, with a previously determined antibody titer, was used on each plate in order to provide a reference to which all sera were standardized, minimizing plate-to-plate and day-to-day variations. The results of these assays, comparing the PorB-PRP conjugate (with and without alum) with conjugates constructed from tetanus toxoid, CRM, are shown in FIGS. 4 and 5A-5G.

Example 5 Comparisons of Hib-rPorB, Hib-TT and Two Commercially Available Hib Vaccines

[0053] Immuno-stimulatory effects of two preparations of the Hib-rPorB conjugate (Hib-rPorB-1 and Hib-rPorB-2), the Hib-TT (tetanus toxoid) conjugate, and two commercially available vaccines, HbOC from Lederle Laboratories, Division of American Cyanamide Company, Pearl River, N.Y., (CRM carrier), and PRP-T from Connaught Laboratories, Inc., Swiftwater, Pa. (tetanus toxoid carrier), were compared.

[0054] Rats (4-6 weeks old) were immunized with conjugated PRP doses of 10 μg at 1, 28 and 49 days. In addition to pre-immune samples, serum samples were taken at 28, 38, 49 and 59 days. Results are shown in FIGS. 6A and 6B. The ELISA IgG titer refers to anti-polysaccharide antibodies.

[0055] The graphic depiction of the data in FIG. 6A shows that the Hib-rPorB conjugates produced a response at least two orders of magnitude greater than that of the other conjugate vaccines. FIG. 6B shows corresponding tabulated data. “Responders” are defined as exhibiting IgG ELISA titers greater than or equal to 4-fold above pre-immune, where all pre-immune values were <50 and adjusted to 25 for calculations. Similar experiments comparing Hib-TT, Hib-rPorB-1, and Hib-rPorB-2 were performed in mice using conjugate dosages of 5.0 μg and 0.5 μg. The data is shown in FIG. 7.

[0056] Finally, eight different preparations of Hib-rPorB (A-H in FIG. 8) were compared to Hib-TT and Hib-CRM conjugates. Hib-rPorB preparations were consistently two orders of magnitude more stimulatory than the Hib-TT or Hib-CRM conjugates, as shown by ELISA assay of anti-polysaccharide IgG antibodies in rats. This data is shown in FIG. 8.

[0057] Having now fully described this invention, it will be understood by those of ordinary skill in the art that the invention can be practiced within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.

References

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1. An H. influenzae type b (Hib) polysaccharide-substantially pure, refolded meningococcal outer membrane protein (rPorB) conjugate.
 2. The conjugate of claim 1, wherein said polysaccharide has a molecular weight range of 5,000 to 50,000.
 3. The conjugate of claim 1, wherein said conjugate is obtained by reductive animation of a Hib polysaccharide and rPorB, wherein the Hib polysaccharide has be oxidized or selectively hydrolyzed to give aldehyde groups.
 4. The conjugate of claim 1, wherein said conjugate is obtained by reductive animation of a Hib polysaccharide and rPorB, wherein the Hib polysaccharide has be oxidized to give aldehyde groups.
 5. The method of any one of claims 1-4, wherein said rPorB is a class 3 rPorB.
 6. A method of preparing an Hib polysaccharide-rPorB conjugate, comprising (a) obtaining an Hib polysaccharide; (b) oxidizing or selectively hydrolyzing said polysaccharide to generate aldehyde groups; (c) obtaining a rPorB; and (d) conjugating the polysaccharide containing aldehyde groups to the rPorB by reductive amination.
 7. The method of claim 6, wherein said Hib polysaccharide is oxidized and has a molecular weight range of 5,000 to 50,000.
 8. The method of any one of claims 6-7, wherein said rPorB is a class 3 rPorB.
 9. The conjugate obtained according to the method of claim
 6. 10. The conjugate obtained according to the method of claim
 8. 11. A pharmaceutical composition comprising the conjugate of claim 1 or 9 and a pharmaceutically acceptable carrier.
 12. A pharmaceutical composition comprising the conjugate of claim 10 and a pharmaceutically acceptable carrier.
 13. A method of inducing an immune response in an animal to H. influenzae, comprising administering the conjugate of claim 1 or 9 to the animal in an amount effective to induce said immune response.
 14. A method of inducing an immune response in an animal to H. influenzae, comprising administering the conjugate of claim 10 to the animal in an amount effective to induce said immune response.
 15. The method of claim 13, wherein said conjugate is obtained by reductive animation of a Hib polysaccharide and rPorB, wherein the Hib polysaccharide has been oxidized to give aldehyde groups.
 16. The method of claim 14, wherein said conjugate is obtained by reductive animation of a Hib polysaccharide and rPorB, wherein the Hib polysaccharide has been oxidized to give aldehyde groups.
 17. The method of claim 13, wherein said polysaccharide has a molecular weight range of 5,000 to 50,000.
 18. The method of claim 14, wherein said polysaccharide has a molecular weight range of 5,000 to 50,000.
 19. The method of claim 13, wherein said rPorB is a class 3 rPorB. 